The present invention relates to compounds useful as analgesic agents. Particularly the compounds of this invention are enkephalin-like compounds. More particular, the invention compounds are structurally related to Met-enkephalin which has been characterized as an endogenous opioid peptide.
Peptides with opiate properties, in particularly the pentapeptides methionine enkephalin (Met-Ek) and (Leu-Ek), have been isolated from extracts of animal tissue and their amino acid sequences have been confirmed [Hughes, J. et al., Nature 258, 577 (1975)]; the biosynthetic pathway for these enkephalins, however, has not been established.
Furthermore, Met-EK and Leu-EK from mammolian brain produce a weak and short lined anagesia following intracerebraventricular or intravenous administration to mice [Buscher, H. H., et al. Nature 261, 423 (1976)] and rats [Belluggi, J. D., et al. Nature 260; 625 (1976)]. The short half life of the activity of the enkephalin may be due to rapid destruction by brain enzymes [N. Marks, et al., Biochem, Biophys. Res. Comm. 74, 1552 (1977)].
Synthetic enkephalin analogs in which Gly.sup.2 has been replaced by D-Ala [Pert, C. B., et al. Science 194, 330 (1976)]; or Leu.sup.5 [Baxter, M. G., et al. Proceed. Brit. Pharm. Soc. p. 455 (1977)] or derivating of enkephalin obtained by increased chain length as H-Tyr-Tyr-Gly-Gly-Phe-Leu-OH or H-Tyr-Gly-Gly-Phe-Leu-Leu-OH [Terenius, L. et al. Biochem, Biophys. Res. Comm. 73, 632 (1976)]. While U.S. Pat. No. 4,103,005 describes the synthesis and biological activity of enkephalin analogs, D-Met.sup.2, Thz.sup.5 and D-Thr.sup.2, Thz.sup.5 -enkephalinamide show prolonged activity and an increased potency. There has been no showing however that these synthetic analogs or derivatives are endogenous opioid peptides.
Our studies to elucidate this enkephalin in biosynthetic pathway were initially centered on the striatum which contained large amounts of enkephalin. This tissue was difficult to work with and difficult to obtain in large quantities. We were thus forced to look for other tissues to work with. Immunocytochemical observations in the adrenal medulla [Schultzberg, M. et al., Neuroscience 3, 1169 (1978)] had shown the presence of relatively large amounts of enkephalin immunoreactive material. Others had shown the presence of enkephalins and possibly other opioid peptides in the adrenal gland. These preliminary studies indicated to us that the adrenal gland might be a suitable tissue in which to study the biosynthesis of the enkephalins. Utilizing high performance liquid chromatography (HPLC) and fluorometric detection we have analyzed the contents of bovine adrenal medullary chromaffin granules. In these granules we have shown the presence of high molecular weight proteins that can be cleaved with trypsin to yield peptides with opioid activity in both radioreceptor and radioimmunoassays. In addition to these large proteins we have found a number of smaller peptides some of which are active in their native form and others that yield active tryptic fragments. We have isolated several of these peptides and precursors and purified some of them to homogeneity.